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>Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity
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Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity
Group A Streptococcus (GAS) (Streptococcus pyogenes) causes more than 700 million human infections each year. The significant morbidity and mortality rates associated with GAS infections are in part a consequence of the ability of this pathogen to coordinately regulate virulence factor expression during infection. RofA-like protein IV (RivR) is a member of the Mga-like family of transcriptional regulators, and previously we reported that RivR negatively regulates transcription of the hasA and grab virulence factor-encoding genes. Here, we determined that RivR inhibits the ability of GAS to survive and to replicate in human blood. To begin to assess the biochemical basis of RivR activity, we investigated its ability to form multimers, which is a characteristic of Mga-like proteins. We found that RivR forms both dimers and a higher-molecular-mass multimer, which we hypothesize is a tetramer. As cysteine residues are known to contribute to the ability of proteins to dimerize, we created a library of expression plasmids in which each of the four cysteines in RivR was converted to serine. While the C68S RivR protein was essentially unaffected in its ability to dimerize, the C32S and C377S proteins were attenuated, while the C470S protein completely lacked the ability to dimerize. Consistent with dimerization being required for regulatory activity, the C470S RivR protein was unable to repress hasA and grab gene expression in a rivR mutant. Thus, multimer formation is a prerequisite for RivR activity, which supports recent data obtained for other Mga-like family members, suggesting a common regulatory mechanism.
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机译：每年，A组链球菌（GAS）（化脓性链球菌）引起7亿多人感染。与GAS感染相关的高发病率和死亡率部分是这种病原体在感染过程中协调调节毒力因子表达的能力的结果。 RofA样蛋白IV（RivR）是Mga样转录调节子家族的成员，以前我们曾报道RivR负调控 hasA 和 grab 毒力的转录因子编码基因。在这里，我们确定RivR抑制GAS存活并在人血中复制的能力。为了开始评估RivR活性的生化基础，我们研究了其形成多聚体的能力，这是Mga样蛋白的特征。我们发现RivR既形成二聚体又形成更高分子量的多聚体，我们假设它是四聚体。由于已知半胱氨酸残基有助于蛋白质二聚化，因此我们创建了表达质粒文库，其中RivR中的四个半胱氨酸均转化为丝氨酸。尽管C68S RivR蛋白的二聚能力基本不受影响，但C32S和C377S蛋白却减弱了，而C470S蛋白完全缺乏二聚能力。与调节活性所需的二聚化一致，C470S RivR蛋白无法抑制 rivR 突变体中的 hasA 和 grab 基因表达。因此，多聚体的形成是RivR活性的前提，RivR活性支持了从其他Mga样家族成员获得的最新数据，表明了一种共同的调控机制。
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